This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. An approach pioneered by Prof. Peter Schultz (Scripps) allows for the site-specific incorporation of an unnatural amino acid anywhere within a protein sequence. This technology has proven useful for labeling proteins with fluorescent, 13C and 15N, and chemical-crosslinking unnatural amino acids for in vivo and in vitro structure-function studies. By using an orthogonal M. jannaschii tRNA/tRNA-synthetase pair, i.e., one where neither the tRNA nor the synthetase cross-reacts with the endogenous E.coli tRNA's or amino acyl tRNA synthetases, it is possible to introduce an unnatural amino acid residue at any position in the protein using a codon that is only recognized by the orthogonal tRNA. The Ortiz de Montellano lab acquired this labeling technology from the Schultz lab and is using it to label heme containing proteins for biophysical and biochemical studies. Information on the extent of incorporation of the unnatural amino acid and the relative expression level of the protein can severely limit the application of this technology. For that reason, we utilize the UCSF Mass Spectrometry Facility to determine the extent and site of incorporation of the unnatural amino acids into the proteins, and to determine their relative expression levels. This knowledge is not only critical for our work, but will make possible the more widespread adoption of this powerful technology.